GIPR Internalization – 2 Color Internalization and Trafficking Assay

GIPR,  the gastric inhibitory polypeptide receptor also known as the glucose-dependent insulinotropic polypeptide receptor is a protein that in humans is encoded by the GIPR gene.  It is a seven-transmembrane protein found on beta cells in the pancrease.

  SpectraGenetics has developed a 2 color  assay that directly measures GIPR internalization, trafficking, and recycling back to the membrane using fluorogen activating peptide technology.

Fluorogen Activating Peptide (FAP) technology combines a genetic tag (to tag the target GPCR), and a fluorogenic dye that only gives signal when bound to the tag.

Using the FAP and cell-impermeant dyes you can selectively label cell-surface and endocytosed protein pools in the following wash-free protocol.

GIPR web

While the confocal images above confirm that the localization of the Green and Red signal to surface and endocytic pools respectively, the signal produced in the assay is homogeneous, so imaging is not required.  The plots below show the assay run on a BD FACS-Vantage cytometer, and are plots of the average fluorescence intensity from the red and green channels from 10,000 events per data point.

GIPR dose

The  panel above shows a dose response of the receptor agonist GIP.  Increasing concentrations of agonist move the cells from predominantly surface GPCR  localization (labeled in Green after the color-exchange), to predominantly endocytosed GPCR (the Red fluorogen, protected from exchange within the endosome).

While it is possible to simply measure an increase in the Red channel to follow agonist-driven endocytosis, measuring the Green channel simultaneously allows you to take the Red/Green ratio or Green-Red difference to obtain a larger assay window.

The same cell line can also assay antagonist response or see internalized receptor return to the surface (re-sensitization) by following these steps:

  1. Add agonist plus red fluorogen and allow time for response
  2. Add green fluorogen
  3. Add test compound (antagonist) and allow time for response
  4. Read both red and green signal


The following products were used to obtain the data in this report:

GIPR Products
We also offer this assay as a service through our partner Sharp Edge Labs ( has tagged more than 150 GPCRs and has validated more than 30 GPCR cell lines.

This report is available as a pdf.